Project Abstract Hematopoietic stem cells (HSCs) reside in the regulatory microenvironment within the bone marrow (BM), termed the niche. Despite extensive research efforts made over the past decades, cellular constituents and locations of the HSC niche have remained controversial. While early studies suggested that HSCs are adjacent to osteoblasts on the bone surface, a growing amount of more recent evidence indicate that HSCs are rather adjacent to sinusoidal vessels in the central marrow; some other showed HSCs are near BM arteries. These conflicting observations may be explained by the heterogeneity of HSCs and the niche, as well as the paucity of markers to identify HSC subsets in the histological analysis. This project focuses on proposing most primitive HSCs among others, and their niche. The overall hypothesis to be tested is that nitric oxide (NO) identifies unprecedented HSCs with distinctly high self-renewing and reconstituting potential. We further hypothesize that these NO+ HSCs are regulated by unique transitional vessels which connect arteries to sinusoidal vessels, while NO- HSCs with a relatively limited self-renewing potential are adjacent to sinusoidal veins. This hypothesis is based on the following preliminary data. We showed that, within CD150+CD48+cKit+Sca1+Lin- HSCs, there was a quiescent population (5-10%) with high expression levels of NO and CD200 receptors (CD200R). These NO+CD200Rhigh HSCs exhibited high blood reconstituting potential which was further increased by serial transplant, while NO- HSCs' reconstituting potential was relatively limited and abolished by serial transplant. This observation suggest NO+ HSCs possess a distinctly high self-renewal and reconstituting potential. Moreover, NO+ HSCs were adjacent to transitional vessels, while NO- HSCs were adjacent to sinusoids which did not express cilia or CD200. To further bring mechanistic insights into NO+ HSCs and their niche and to promote clinical translation of our basic findings, we will pursue the following aims. In Aim 1, we will seek to characterize NO+ HSCs' self-renewal, reconstituting potential and molecular features, and elucidate their intrinsic regulatory mechanisms of NO+ HSCs, by using the combination of single cell RNA sequencing, transplantation assay, genetic deletion, and in vivo microscopy tracking of symmetric or asymmetric division of photolabeled individual HSCs. In Aim 2, we will elucidate NO+ HSCs' locations and extrinsic regulatory mechanisms. We will validate localization of NO+ HSCs to transitional vessels by performing deep whole-mount 2-photon BM imaging of novel HSC-reporter mice. We will further elucidate roles of transitional vessels' CD200 and cilia in HSC regulation and protection from radiation stress. We will finally investigate therapeutic potential of CD200R agonist treatment to mitigate post-irradiation hematopoiesis failure. Completion of the project may address the controversy about the niche location by proposing unprecedented primitive HSCs and its niche. Successful studies will further identify cilia-CD200/CD200R-NO axis as new HSC regulators, leading to novel treatments for hematopoiesis failure.